The P.II or Opa outer membrane proteins of the gonococcus undergo both phase variation (on-off switching) and antigenic variation (expression of different antigenic variants of the protein). There is suggestive evidence that P.II expression is important in gonococcal pathogenesis, although the role of the proteins in infection is still not clear. Expression of opa genes involves novel genetic regulatory mechanisms. This project will focus on questions concerning the importance of P.II in gonococcal virulence and the genetic regulatory mechanisms governing expression and variation of P.II proteins. The first aim is to determine if expression of P.II proteins is required for establishment of uncomplicated mucosal infection. The human challenge studies to be carried out in Project 3 of this application provide an opportunity for a definitive test of this question. Gonococcal mutants unable to express particular P.II proteins will be constructed, using recombinant DNA techniques; infectivity of the mutants and the wild-type parental strain in male volunteers will be compared. The second aim concerns genetic regulatory mechanisms for P.II phase variation, which occurs as a consequence of changes in the number of copies of a repetitive DNA sequence element in the 5' coding sequence of opa genes, most probably by a process of slipped-strand mispairing. Using gene fusions as assay systems for phase variation, the possible role of a histone-like protein in modulating phase variations through effects on DNA topology will be investigated. Additional genetic loci affecting P.II phase variation will be identified, and the effects of environmental conditions likely to be encountered in vivo on the process will be determined. The third aim focuses on mechanisms of recombination among opa genes. Such recombination events generate new combinations of coding sequences and the antigenic determinants they encode, increasing diversity in the Protein II family. Assay systems for recombination, using the polymerase chain reaction or tracking movement of specific DNA sequences among opa loci, have been developed. These assays will be used to determine if opa recombination involves site-specific or generalized recombination, via intrachromosomal recombination or some other mechanism such as genetic transformation. These studies will provide insight into the mechanisms that gonococci use to vary their surface array and enhance survival in the host, and into the role of Protein II as a virulence factor in gonococcal infection.